Cryo-EM structure of ex vivo fibrils associated with extreme AA amyloidosis prevalence in a cat shelter

AA amyloidosis is a systemic disease characterized by deposition of misfolded serum amyloid A protein (SAA) into cross-β amyloid in multiple organs in humans and animals. AA amyloidosis occurs at high SAA serum levels during chronic inflammation. Prion-like transmission was reported as possible cause of extreme AA amyloidosis prevalence in captive animals, e.g. 70% in cheetah and 57–73% in domestic short hair (DSH) cats kept in zoos and shelters, respectively. Herein, we present the 3.3 Å cryo-EM structure of AA amyloid extracted post-mortem from the kidney of a DSH cat with renal failure, deceased in a shelter with extreme disease prevalence. The structure reveals a cross-β architecture assembled from two 76-residue long proto-filaments. Despite >70% sequence homology to mouse and human SAA, the cat SAA variant adopts a distinct amyloid fold. Inclusion of an eight-residue insert unique to feline SAA contributes to increased amyloid stability. The presented feline AA amyloid structure is fully compatible with the 99% identical amino acid sequence of amyloid fragments of captive cheetah.

seed and amyloid template introduced via horizonal transmission has no substrate upon which to act. That said, cat and cheetah SAA structures may be particularly effective amyloid templates and promote disease especially in the conditions of captivity.

Specific comments
Introduction.
Lines 67-68. This sentence could be re-worded. It is a bit dogmatic; while there are data supporting the idea that high levels of SAA lead to impaired lysosomal function and resultant accumulation of oligomers, this has not been conclusively proven.
Line 72. Add "A comparison of" cryo-EM structures.... Line 73. Cryo-EM was performed on ex vivo amyloid subunit proteins, not on amyloid deposits, which contain various other proteins and biomolecules.
Line 83. Define DHS at first usage.
Line 93. This is confusing. Does "deposited structure" refer to an amyloid deposit or something deposited in a database?
Results and Discussion, Figures, and Methods. Figure S1A. Why not provide actual values rather than show data as a bar graph? If these data are to be presented, then state when blood was drawn -at necropsy versus prior to death. The data are probably not necessary. Figure 1. The Congo red is fine, but the immunofluorescence is not informative. There is no mention in the Results, Methods of Figure Legend about ThT. It seems to be masking immunoreactivity. It would be more helpful to see anti-SAA staining in the absence of ThT and preferably via immunoperoxidase detection rather than immunofluorescence. Co-localization between Congo red and immunostaining could be revealed. In the legend, what is meant by "hotspots"?
The SAA antiserum is not well-described. A reference for or description of the virus-like particles needs to be provided. Also provide the cat SAA Uniprot ID used for designing the peptides, as well as the residue numbers which they represent (e.g., residues 10 -20). Amyloid is notoriously sticky when it comes to binding antibodies. To make the staining more convincing, show an image in which primary antibody is left out.
Line 214. 1st sentence of Methods states that full details were presented in Reference 47. Reference 47 is in bioRxiv and is a preprint of this exact manuscript and provides no further information. This needs to be corrected.
A couple of other points: 1. SAA is a serum protein. The protein comprising SAA-derived amyloid is termed AA protein.
2. The numbering system used in the manuscript is bothersome. SAA, the protein found in the circulation, lacks a signal peptide; the N-terminus of secreted, mature SAA is most commonly denoted as residue 1, regardless of the species. 3. Show (in Supplementary Material) a denaturing gel electrophoresis picture (Coomassie blue-stained SDS-PAGE) of the preparation on which cryo-EM was performed. 4. Expand the description of the procedure used to solubilize the extracted fibrils. It simply states that they were dissolved in 8 M urea. At what temperature and for how long was this done; this is important since urea can induce chemical modifications. What is meant by "1/6 diluted" (% w/v or 1 part 8 M urea, 6 parts ammonium bicarbonate?). 5. Provide some LC-MS/MS data. Table S1 simply lists Uniprot entries. Show which peptides were detected and their relative abundance and/or show the mass of the intact AA protein. This is important because it is stated that the AA protein analyzed was 76 residues, yet there are no data supporting this.
Line 155. References for human and mouse structures are needed.
Reviewer #3 (Remarks to the Author): The language could be a little simpler in places, e.g. "their molecular identities define specific disease forms and organ distribution" P4/L51 -does this mean 'different amyloid proteins aggregate in different places and cause different diseases". A simpler more direct use of language might help the general reader. P5/L51 -"highly polymorphic structures". I think this a slight misuse of language. I believe the authors mean that the mouse and human structures in Liberta et al 2019 are different -I agree. Saying that they are polymorphic suggests (to me) that each of the human and mouse samples were polymorphic (i.e. many different structures), which was not the case I believe.
P5/L86 -I think parenteral will not be well understood by the general reader without diving into the supporting literature. An extra few words of explanation would help the MS at this point. Figure 1 seems a little superfluous to me. The structural biology is generally fine, and I have only a few relatively minor questions on the MS or the presentation of the structural results.
1. I would ask the std cryoEM reviewer's question though. What is in the fibril segments that don't make it into their final reconstruction -they state the fibrils are homogeneous (P6/L118), but then only use 17% of their data -a sentence of two and a supp figure of their classification scheme would help here. 2. Re their point about the cis proline, I would guess they are correct, but I'm not 100% convinced by their figure at this resolution -a it is 'consistent with' a cis conformation but not definitive -as they themselves suggest in the methods. I think this is uncertain at this stage. 3. They state that the fibrils are left-handed but how was this determined? There's no evidence presented to support an experimental determination of this?
In general the MS is clear and well written, and presents a novel, interesting structure. However, I think it falls short of the standard for Nature Communications. The authors state that this is, 'the first ex vivo structure of a spontaneously occurring amyloid from an animal kept in a man-made habitat', which is true but of relatively minor impact in the field. The big interest would be the prion-like nature of the spread, but I cannot see anything in the structure or their description of it, that informs on the properties of this fibril that might contribute to that phenomenon. Without this, it is 'just' a solid piece of structural biology.
Overall therefore, I feel it falls short of the 'in field impact' required at Nature Comms.

Response to Reviewers
Responses are organized as follows: #1) raised point >>> response and explanation to reviewer p. 5/ ll. 11: page / line numbers and relevant text changes/ highlighted in yellow in the text ### Reviewer #1: #1.1) In this interesting and novel study, Rigano and colleagues report a new 3.3A resolution cryo-EM structure of ex vivo amyloid fibrils from a cat with amyloid A (AA) amyloidosis. The fibril fold differs from those observed previously in ex vivo human and murine AA fibrils, thus contributing to the rapidly growing repertoire of amyloid folds.
The authors report Pro cis-isomer in cat SAA (cSAA) fibrils, which has not been previously reported in amyloids. The study is well performed, technically sound and well written. Comments below relate mainly to the interpretation of results.
>>> We are grateful to the positive evaluation by reviewer #1.

#1.2) Pro in a cis conformation in amyloid is an interesting observation. This Pro is
100% evolutionarily conserved in the SAA protein family and forms a tight turn between helices 2 and 3 in the native fold. Since Pro isomerization is a slow step in protein misfolding, the authors may consider whether the presence of Pro in a cis conformation in both native and fibrillary SAA might relate to the apparent ease of AA transmissibility in cats.
>>> Pro-66 adopts trans conformation in native lipid-free human SAA (PDB id: 4IP8) and retinol-bound mouse SAA (PDB id: 6PY0). However, the required unfolding during the α-to-β transition of native SAA to AA amyloid may allow for a spontaneous conversion from the trans-to the nearly isoenergetic cis-isomer, but could also be catalyzed by extracellular matrix (ECM) localized Peptidyl prolyl cis/trans isomerases  Figure   S3. We speculate that the cis-Proline induced re-orientation of the C-terminal meandering tail may allow feline AA amyloid to adopt a thermodynamically more stable structure, not accessible with trans-Pro-66.

#1.3)
The authors should be careful relating fibril structure to the apparent prion-like transmissibility; the latter may be mediated by oligomeric intermediates (of unknown structure) rather than fibrils.
>>> We included specific information about non-fibrilllar and fibrillar fragments of prions in the introduction. Regarding transmissibility of AA amyloidosis, we note that Zhang and co-workers 4 have demonstrated that AA amyloid extracted from the feces and liver of cheetah worked as amyloid enhancing factor when administered to pre-inflamed mice. However, we have used careful language to indicate the uncertainties of our study, and of claims within the research field. p. 5/ ll. 89: The classical prion disease transmissible spongiform … p. 11/ ll. 213: Whether auto-catalytic, self-perpetuating and …

#1.4)
The fibrils were extracted from kidney, but the cat apparently also had liver amyloidosis. Is the fibril structure (or at least the gross fibril morphology) similar in the kidney and liver of this cat? If this analysis has not been performed, then the comparison between cat and cheetah amyloid fibril structure should be definitely toned down. Based on the a.a. sequence similarity between cat and cheetah SAA, the authors propose similar AA amyloid structure in these animals. This is a stretch given the extreme sensitivity of the amyloid fold to the environmental conditions and conservative residue substitutions.
The authors correctly state that the two a.a. substitutions that differentiate these feline species are compatible with the cat fibril structure reported here. They should probably stop at that given lack of further evidence.
>>> The reviewer has raised important points regarding polymorphism of amyloids across organs, and in this case between related species. We absolutely agree that these are important questions that we aim to address in future studies. At this stage we have decided to follow the reviewer's advice to tone down our speculative statement about similar or identical structures. >>> we thank the reviewer for pointing us to these interesting studies. We included two new references and changed the sentence accordingly. >>> no comment required.

#2.2)
The overriding theme is that cat AA amyloidosis develops and progresses via a prion-driven mechanism. Nowhere do the authors explain what they mean by priondriven and prion capacity. A brief explanation needs to be provided in the Introduction.
>>> We agree that prion transmission has not been introduced properly which is crucial to put the structural findings in context. >>> We added missing information about the Thioflavin S staining in materials&methods, and the legend of Figure S1, as well as in the Results section. We removed the term hotspots. Herein we also refer to the more detailed M&Ms of the related manuscript. p. 7/ ll. 123: … of Thioflavin-positive amyloids in all three organs p.12/ ll. 237: … were stained with 1% (w/V) Thioflavine S and DAPI, respectively. >>> We also thank the reviewer for pointing out apparent masking effects in the composite image, not revealing any co-localization. However, the lack of co-localization was mainly due to non-optimal presentation of our existing imaging data. To facilitate interpretation of the staining, we added images of the separately acquired Thioflavin S (green) and anti-SAA (magenta) stainings as separate panels in Supplementary Figure 1.
Furthermore, we noticed non-matching relative intensity profiles in the two channels of the digitally merged composite image, which have been carefully matched in the new version. Furthermore we substituted red by magenta to generate color-blind friendly figures. In the new composite images, co-localized staining at matched relative signal intensity appears white. Attached to this report we also provide the raw grey-scale 16 bit images of the separate channels, as well as secondary antibody control images that were generated for organs of other cats presented in our second study (Attachments 1 and 2). Figure S1, what is meant by "hotspots"? >>> Indeed, this term is misleading and was deleted.

#2.5.5)
The SAA antiserum is not well described. A reference for or description of the virus-like particles needs to be provided. Also provide the cat SAA Uniprot ID used for designing the peptides, as well as the residue numbers which they represent (e.g., residues 10 -20). Amyloid is notoriously sticky when it comes to binding antibodies. We have added the Uniprot ID, added a new reference for the virus-like particles, and also refer to our related study. >>> We believe to have followed this definition except for the paragraph "Cat´s distinct AA amyloid structure buries its unique eight-residue insert between the two protofilaments and is predicted as the most stable assembly". We have changed the abbreviation of the human, mouse and cat AA amyloid structures to hAA, mAA and cAA.

>>>
p. 9/ ll. 172: Compared to the 54-residue … #2.6.2) The numbering system used in the manuscript is bothersome. SAA, the protein found in the circulation, lacks a signal peptide; the N-terminus of secreted, mature SAA is most commonly denoted as residue 1, regardless of the species.
>>> The reviewer has pointed to a general problem that residue numbers of many PDB-   >>> We have chosen to present the complete case study from disease diagnosis to molecular structure. We believe it may be of interest for readers to have a full view on these amyloids from the tissue of origin to the structure.  Herein, we provide further information on the initial 3D classification after the first 3D auto-refinement with particle numbers of each class:

#3.5)
Evidently, three initially "good" looking classes sum up to 335k particles, thus about 88% of the initially picked particles. The following iterative Bayesian reconstruction selects particle subsets based on optimal particle alignment, for best resolution.

#3.5.2)
Re their point about the cis proline, I would guess they are correct, but I'm not 100% convinced by their figure at this resolution -a it is 'consistent with' a cis conformation but not definitive -as they themselves suggest in the methods. I think this is uncertain at this stage. p. 14/ ll. 302: … we modeled Proline-66 as cis-isomer to fit the backbone carbonyl into the map, although a higher resolution is required to discriminate conclusively between cis-and trans-Proline.

#3.5.3)
They state that the fibrils are left-handed but how was this determined? There's no evidence presented to support an experimental determination of this?
>>> The initial map was actually right-hand twisted, but we failed to build a model with good statistics into this map. We could fix these issues by building the model into the inverted map. We also note that the vast majority of amyloids is left-handed. Therefore we concluded that this particular amyloid is very likely left-handed. The information about the model re-building into the inverted map was already described in our previous version, and is now found here: p. 14/ ll. 299: Molprobity validation revealed model issues that were resolved by rebuilding of a single chain into the inverted map with left-handed twist.

#3.5.4)
In general the MS is clear and well written, and presents a novel, interesting structure.
>>> We thank the Referee for this comment.

#3.5.5)
However, I think it falls short of the standard for Nature Communications. The authors state that this is, 'the first ex vivo structure of a spontaneously occurring amyloid from an animal kept in a man-made habitat', which is true but of relatively minor impact in the field. The big interest would be the prion-like nature of the spread, but I cannot see anything in the structure or their description of it, that informs on the properties of this fibril that might contribute to that phenomenon. Without this, it is 'just' a solid piece of structural biology. Overall therefore, I feel it falls short of the 'in field impact' required at Nature Comms.
>>> Obviously, we disagree. The presented novel feline AA amyloid fold comes from a very specific environment in which AA amyloidosis, a rare disease, is strikingly common.
The data and analysis shown in this manuscript also helps the field to contextualize previous observations of prion-like transmission in captive cheetah. In particular, we have put forward novel testable hypotheses about increased prion propensity of the feline SAA variant. Inclusion of the feline-specific eight-residue insert in the amyloid core increases amyloid core mass and stability (Supplementary Figure 9, also comprising additional solvation free energy estimates), which likely contributes to increased environmental persistence and clearance resistance in the host. We believe that our data will inspire other researchers in the field to design new experiments to test our hypothesis. We also want to comment on the prion-related statement that the reviewer 'cannot see anything in the structure or their description of it, that informs on the properties of this fibril that might contribute to that phenomenon.  General comments:

>>>
In addressing the issues raised in the first reviews, the authors have packed in more references and figures, but have not really enhanced the paper. It seems that because the paper has been submitted to an online-only journal, there has been less regard to maintain succinctness. At times the text is rambling, compromising the focus of the manuscript. The 111 references, some of which date back to 1972, are excessive in number and duplicative; in addition, reference formatting is inconsistent, and in some cases (e.g., the journal Amyloid), the name of the journal is incorrectly noted. While this may not be a substantive point, it is annoying as is the repeated use of the "amyloids" instead of simply amyloid and the use of "cat's SAA and mouse's SAA" instead of cat and mouse SAA. Somewhere in the middle of the Results, the authors start using the abbreviations cSAA, mSAA and hSAA for cat, mouse and human SAAs -why not use the abbreviations at the outset? These issues exemplify a pervasive lack of attention.

REVIEWER COMMENTS
Reviewer #2 (Remarks to the Author): As noted in the original review, the comments of this reviewer are not an evaluation of the cryo EM results or their structural interpretation.
Specific comments: #1.1) Figure 1. The histology showing Congo red staining is fine and should be included; it confirms the presence of amyloid. The immunofluorescence and thioflavin S staining are no more informative than they were in the original submission. There is no control to verify antiserum specificity or to demonstrate the extent of background thioflavin S staining in amyloid-negative tissue. The authors could simply omit Figure  1B. >>> Herein, we address the two related points. We have removed Figure 1B   We can only speculate that the "missing" residues are simply too flexible/disordered to be observed in the map. It is very common that part of the polypeptide is invisible in Cryo-EM structures of amyloid fibrils. >>> The correct id is listed. The dataset has not officially been released. As we have already written in our last response, the reviewer can access the data using following login details: website: https://www.ebi.ac.uk/pride/login Username: reviewer_pxd035851@ebi.ac.uk Password: OnCWq1fK

#1.4) General comments:
In addressing the issues raised in the first reviews, the authors have packed in more references and figures, but have not really enhanced the paper. It seems that because the paper has been submitted to an online-only journal, there has been less regard to maintain succinctness. At times the text is rambling, compromising the focus of the manuscript. The 111 references, some of which date back to 1972, are excessive in number and duplicative; in addition, reference formatting is inconsistent, and in some cases (e.g., the journal Amyloid), the name of the journal is incorrectly noted. While this may not be a substantive point, it is annoying as is the repeated use of the "amyloids" instead of simply amyloid and the use of "cat's SAA and mouse's SAA" instead of cat and mouse SAA. Somewhere in the middle of the Results, the authors start using the abbreviations cSAA, mSAA and hSAA for cat, mouse and human SAAs -why not use the abbreviations at the outset? These issues exemplify a pervasive lack of attention.
>>> We strongly disagree with the overall assessment by Referee 2 but for sake of brevity here we list only the modifications and amendments compatible with the above comments.
We have removed some "duplicative" citations to stay below 100 references in total. While about 55-60 of these citations are related to the scientific background, the additional ~40 citations are referring to methodological/technical work essential for us to obtain and analyze our work, therefore deserving credit. We have also removed the abbreviations, since they were only used in a single paragraph.